Hepatitis C virus (HCV) infection is found in 0.5% to 8.0% of blood donors worldwide. Because the infection is chronic in more than 60% of infected persons, the disease is an important public health and economic problem. The management of patients with chronic hepatitis C is complex--the disease is often only mildly symptomatic and slowly progressive, but 20% of patients develop cirrhosis after 20 years of infection and perhaps 10% of those with cirrhosis develop hepatocellular carcinoma. It is also an important indication for liver transplantation. In Europe and Japan the disease is more important numerically than is either hepatitis B or HIV infection. Existing antiviral agents are effective in only a minority of patients, yet good responses can be obtained.
An important target for the treatment of HCV is nonstructural protein 3, a protease encoded by HCV. This NS3 protease associated with human hepatitis C virus is an unstable protein in the absence of high concentrations of detergent. To stabilize the NS3 protease to sufficient quantities for biochemical, kinetic, and biophysical analyses, as well as for the construction of antiviral screening assays, ionic or non-ionic detergents need be incorporated both during purification and analyses. Antiviral leads discovered with detergent treated NS3 protein are not useful. Further, the presence of high quantities of detergents renders significant difficulties in the precise interpretations of biochemical, kinetic, and biophysical analyses. In some cases (e.g., sedimentation, protein crystallization), the presence of detergents preclude biochemical, kinetic, and biophysical analyses.
Prior methods employed detergents and glycerol for purification of NS3. Applicants have discovered a method of purifying NS3 without detergent, with from 5% to about 20% glycerol, preferably 7-12% and with high stability and activity. The resultant enzyme displays a higher catalytic activity than what is known for this protease, that is, 10-500 fold more active than the prior art preparations, depending on the form of the enzyme. Purification according to the methods of the present invention ensures a high stability of the NS3 protease, rendering it amenable to kinetic, biochemical, and biophysical analyses in the absence of detergents. Prior art methods do not afford a stable, detergent free NS3 for enzymologic, biochemical, and biophysical studies.
When properly expressed and prepared from the cloned plasmid in E. coli, the NS3 protease is obtained in milligram quantities in the complete absence of detergent. The resultant enzyme is very soluble and stable for long periods of time (weeks to months at 4.degree. C. and &gt;12 months at -80.degree. C.), and displays high catalytic activity.
An assay with the detergent free HCV NS3 protease is useful as a screening tool for HCV antivirals as well as a diagnostic tool for diseases resulting from HCV infection. The potency range of the HCV antivirals can range from subnanomolar to micromolar concentrations.